Targeting tumor microenvironments with recombinant prokaryotic collagen-like proteins

ABSTRACT

A recombinant collagen-like protein comprising a binding domain having binding capacity for both extra domain A and extra domain B-containing variants of cellular fibronectin. Cancer may be treated BY administering the recombinant collagen-like protein to a patient.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under NSF Award Number: DGE1144676 awarded by the National Science Foundation. The government has certain rights in the invention.

TECHNICAL FIELD

Various embodiments disclosed herein relate generally to various recombinant prokaryotic collagen-like or triple helical proteins, methods of manufacture and use.

BACKGROUND

The concept of drug delivery to tumors holds promise for improved therapeutics, enabling specific delivery of drugs and biologics to pathological tissue, thereby avoiding off-target effects, and reducing the toxicity of currently used therapies. To target these sites, drugs are conjugated to carriers that recognize the tumor microenvironment, such as overexpressed cell-surface and secreted markers.

However, current cancer biomarkers still pose a significant off-target risk due to their expression on healthy cells.

Collagens are major structural proteins present in the extracellular matrix (ECM) of animals. They are defined by a characteristic triple-helix structure that requires a (Gly-Xaa-Yaa), repeating sequence. The residues found in the Xaa and Yaa positions are commonly proline, where Pro in the Yaa position in mammalian collagens is post-translationally modified to hydroxyproline (Hyp) which enhances helical stability. Prokaryotic collagen-like proteins form stable triple helices without the presence of hydroxyprolines and have been shown to be expressed in several bacteria, including Streptococcus pyogenes and filaments on Bacillus anthracis spores.

Fibronectin is a high molecular weight glycoprotein of the ECM and is found throughout the human body in two main forms. Plasma fibronectin, which is produced by hepatocytes in the liver, is secreted in a soluble form into the blood stream, whereas cellular fibronectin (cFn), is produced by a variety of cells, including fibroblasts, and is deposited as an insoluble crosslinked protein in tissues. Humans express over 20 cFn isoforms due to alternative splicing of a single fibronectin gene, FN1, that differ from plasma fibronectin by the inclusion of additional fibronectin type III repeats, extra domains A (EDA/EIIIA), B (EDB/EIIIB) and the variable connecting segments (VCS). Specifically, EDA/cFn and EDB/cFn are expressed during embryogenesis but only at negligent levels in normal adult tissue. However, the expression of both EDA/cFn and EDB/cFn isoforms is substantially upregulated within the tumor microenvironment. Similarly, Tenascin-C (TNC) is a related ECM protein that contains fibronectin type III repeats and has a similar expression pattern to EDA/cFn and EDB/cFn. Thus, EDA- and EDB-containing cFn isoforms, as well as TNC, are attractive biomarkers for targeting the tumor microenvironment.

Antibodies that are able to target EDA and EDB cFn isoforms have been designed, wherein such antibodies are conjugated with pro-inflammatory cytokines to illicit an immune response against cancerous cells. Antibodies, aptamers and small immunoreactive proteins (SIPs) have also been designed to target TNC in tumors. However, antibodies are mono-specific and cannot target more than one tumor-associated ligand at the same time. Additionally, antibody production is costly and time-consuming.

The streptococcal collagen-like protein-1 (Scl1) is a ubiquitous surface adhesin, which is co-expressed with a range of known virulence factors that are regulated by the multiple virulence gene regulator of Group A Streptococcus (GAS). Scl1 is a homotrimeric protein protruding from the GAS surface that contains four structurally distinct regions. The outermost N-terminal variable (V) region is adjacent to a collagen-like (CL) region that consists of a varying number of GlyXaaYaa (GXY) repeats and adopts stable collagen-like triple helices. At the C-terminus, Scl1 contains a linker (L) region which is a series of conserved, direct repeats adjoining the CL region to the cell wall/membrane (WM)-associated region. Functionally, Scl1 has been shown to bind host-cell integrin receptors and plasma components. Scl1 has also been shown to bind to cellular fibronectin, but not plasma fibronectin. Scl1 is also recognized to play a significant role in biofilm formation on abiotic surfaces.

The inventors have discovered that recombinant proteins derived from bacterial collagen-like proteins, such as Scl1, can serve as effective tools for targeting the tumor microenvironment.

SUMMARY OF EXEMPLARY EMBODIMENTS

Various embodiments recite a recombinant collagen-like protein, wherein the protein includes a binding domain having the capacity to bind to both extra domain A and extra domain B-containing variants of cellular fibronectin. In various embodiments, the collagen-like protein is a prokaryotic collagen-like protein, such as a streptococcal collagen-like protein from a Group A Streptococcus. In various embodiments, the Group A Streptococcus is Streptococcus pyogenes. In various embodiments, the streptococcal collagen-like protein is a variant of Scl1 or Scl2 of S. pyogenes or a combination thereof.

Various embodiments also recite a recombinant collagen-like protein further having a capacity to bind to Tenascin-C.

Various embodiments also recite a recombinant collagen-like protein, wherein the collagen-like protein further recognizes collagen-binding integrin receptors α₂β₁ and α₁₁β₁.

Various embodiments recite a recombinant collagen-like protein having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

Various embodiments also recite an expression vector including a nucleotide sequence encoding the recombinant collagen-like protein.

Various embodiments also recite a host cell including an expression vector including a nucleotide sequence encoding the recombinant collagen-like protein.

Various embodiments recite pharmaceutical compositions including a recombinant collagen-like protein, wherein the protein includes a binding domain having the capacity to bind to both extra domain A and extra domain B-containing variants of cellular fibronectin.

Various embodiments also recite a method of treating cancer that involves administering a recombinant collagen-like protein, wherein the protein includes a binding domain having a capacity to bind to both extra domain A and extra domain B-containing variants of cellular fibronectin.

Various embodiments also recite a method of treating cancer that involves administering a recombinant collage-like protein having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

Various embodiments also recite a method of preparing a collagen-like protein including a binding domain having the capacity to bind to both extra domain A and extra domain B-containing variants of cellular fibronectin that involves combining (i) two copies of an integrin recognition sequence GLPGER inserted into the collagen-like (CL) domain of a protein of SEQ ID NO: 4; and (ii) the variable (V) domain from a protein of SEQ ID NO: 3.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand various embodiments, reference is made to the accompanying drawings, wherein:

FIG. 1 illustrates a model of rScl hybrid constructs as targeting agents to the tumor microenvironment.

FIGS. 2A and 2B illustrate the binding of rScl hybrid constructs to recombinant rEDA, rEDB and Tenascin-C.

FIG. 3 illustrates the structure of rScl hybrid constructs 1 and 2.

To facilitate understanding, identical reference numerals have been used to designate elements having substantially the same or similar structure or substantially the same or similar function.

DETAILED DESCRIPTION

The description and drawings presented herein illustrate various principles. It will be appreciated that those skilled in the art will be able to devise various arrangements that, although not explicitly described or shown herein, embody these principles and are included within the scope of this disclosure. As used herein, the term, “or” refers to a non-exclusive or (i.e., and/or), unless otherwise indicated (e.g., “or else” or “or in the alternative”). Additionally, the various embodiments described herein are not necessarily mutually exclusive and may be combined to produce additional embodiments that incorporate the principles described herein.

This disclosure relates to defined isoforms of the extracellular matrix (ECM) protein, cellular fibronectin (cFn) and Tenascin-C (TNC) as biomarkers for the delivery of drugs to the tumor microenvironment. This disclosure further relates to recombinant proteins derived from bacterial collagen-like proteins that may serve as effective tools for targeting the tumor microenvironment

In some embodiments, the bacterial collagen-like protein is derived from a prokaryotic collagen-like protein, such as a Streptococcal collagen-like protein. In some embodiments, the Streptococcal collagen-like protein is selected from a group that includes an Scl1 or Scl2 protein, a variant of an Scl1 or Scl2 protein, or combinations thereof.

In various embodiments, the collagen-like proteins may be designed to have binding capacity for isoforms of the extracellular matrix (ECM) protein, cellular fibronectin (cFn) and Tenascin-C (TNC) that may be found within the tumor microenvironment. In various embodiments, the collagen-like proteins may be designed to have binding capacity for extra-domain A isoforms of cellular fibronectin (EDA/cFn), extra-domain B isoforms of cellular fibronectin (EDB/cFn), TNC isoforms, or combinations thereof.

In various embodiments, the collagen-like proteins may further have binding capacity for integrin receptors. In some embodiments, the collagen-like proteins may be designed to have binding capacity to α₂β₁ and α₁₁β₁ receptors that are upregulated on cancer cells.

In various embodiments, the collagen-like protein may include an rScl1 construct composed of a rod-shaped collagen-like domain (CL) and a globular variable (V) domain, as shown in FIG. 1. In various embodiments, the rScl1 construct may be designed to bind to at least one isoform of cellular fibronectin, selected from the group consisting of EDA/cFn, EDB/cFn and TNC, and promote human cell attachment through collagen receptors, integrins α₂β₁ and α₁₁β₁.

In various embodiments, the collagen-like protein may include a recombinant Scl1 hybrid construct characterized by SEQ ID NO: 1. In another embodiment, the collagen-like protein may include a recombinant Scl1 hybrid construct characterized by SEQ ID NO: 2.

In some embodiments, the recombinant Scl1 constructs may be derived from a parental Scl1 sequence. In certain embodiments, the recombinant Scl1 constructs are derived from parental Scl1 sequences of SEQ ID NOS: 3-5. In one embodiment, the parental Scl sequence rScl1.1 of SEQ ID NO: 3 is derived from the scl1 allele from a wild-type M1 serotype strain of group A Streptococcus (GAS). The amino acid sequence of rScl1.1 is the same as the sequence in the genome of M1, however, the rScl1 proteins lack the C-terminally located Linker and Cell Wall Attachment Domains found in the endogenous protein. In another embodiment, the parental Scl sequence rScl2.28 of SEQ ID NO: 4 is derived from the scl2 allele from a wild-type M28 serotype strain of GAS. The amino acid sequence of rScl2.28 is the same as the sequence in the genome of M28, however, the rScl2 proteins lack the C-terminal Cell Wall Attachment Domain found in the endogenous protein. In another embodiment, the parental Scl sequence rScl1.41 of SEQ ID NO: 5 is derived from the scl1 allele from a wild-type M41 serotype strain of GAS. The amino acid sequence of rScl1.41 is the same as the sequence in the genome of M41, however, the rScl1 proteins lack the C-terminally located Linker and Cell Wall Attachment Domains found in the endogenous protein.

In an exemplary embodiment, the rScl.hybrid1 construct (SEQ ID NO: 1) shown in FIG. 1 may be designed to have two desired biological functions: (i) 2 copies of the integrin recognition sequence ((GLPGER (SEQ ID NO: 6)) placed into the inert CL domain of rScl2.28 (SEQ ID NO: 4); and (ii) the V domain from the TNC and EDA/EDB-binding protein rScl1.1 (SEQ ID NO: 3). The (GLPGER)₂-CL domain was found to promote efficient internalization of group A Streptococcus expressing the rScl1.1 protein, compared to group A Streptococcus that expresses inert rScl2.28. In this embodiment, the V domain of rScl1.1, specifically the loop-region of Scl1.1-V domain, binds to EDA/cFn and to rEDB and TNC. Thus, in this embodiment, the rScl.hybrid1 construct possess both the capacity to bind to TNC, as well as EDA/cFn and EDB/cFn isoforms, highly expressed within and around tumor/cancerous tissues, as well as the capacity to promote internalization via integrin receptors α₂β₁ and α₁₁β₁.

The hybrid Scl1 proteins substantially described in FIG. 1 differ from current TNC and EDA/EDB targeting technologies due to their capacity to target a combination of biomarkers that include TNC, and both of EDA/cFn and EDB/cFn, as well as recognize collagen-binding integrin receptors α₂β₁ and α₁₁β₁, enabling both tissue localization and internalization of conjugated drugs, cytokines, and siRNAs. In contrast, antibody therapies are only able to target 1 ligand per antibody.

In various embodiments, the hybrid Scl1 proteins of the invention may be produced using any suitable expression system, including 6x His-tag expression systems and strep-tag expression systems. In various embodiments, the expression vector comprises a nucleotide sequence encoding the hybrid Scl1 proteins of the invention.

In various embodiments, there is provided a host cell comprising and expressing an expression vector having a nucleotide sequence encoding the hybrid Scl1 proteins of the invention. Suitable host cells include prokaryotic cells, such as Escherichia coli, Streptococcus and Bacillus.

In various embodiments, the collagen-like proteins may be conjugated to a therapeutic agent. In some embodiments, the collagen-like protein may form conjugates with various therapeutic agents used in the treatment of cancer, such as chemotherapeutic agents. Exemplary chemotherapeutic agents include busulfan, improsulfan, piposulfan, benzodepa, carboquone, meturedepa, uredepa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, aclacinomycins, actinomycin F(1), anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carzinophilin, chromomycin, dactinomycin, daunorubicin, daunomycin, 6-diazo-5-oxo-1-norleucine, doxorubicin, epirubicin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, fluororacil, tegafur, L-asparaginase, pulmozyme, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, carboplatin, cisplatin, defofamide, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, etoposide, flutamide, gallium nitrate, hydroxyurea, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, sizofiran, spirogermanium, paclitaxel, tamoxifen, teniposide, tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, urethan, vinblastine, vincristine, and vindesine.

The present disclosure further relates to pharmaceutical compositions containing the recombinant collagen-like proteins of the invention. In some embodiments, the recombinant collagen-like protein may be formulated in admixture with a pharmaceutically acceptable carrier. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In a preferred embodiment, the carrier is a nanoparticle. In a more preferred embodiment, the pharmaceutical composition contains a nanoparticle coated with the recombinant collagen-like protein of the invention.

Another aspect of the present disclosure provides a method of treating cancer that involves administering a therapeutically effective amount of the collagen-like proteins of the invention. In various embodiments, the collagen-like proteins of the invention may be used to treat growing tumors as well as metastatic cancers. Exemplary types of cancer include breast cancer, prostate cancer, melanoma, gastric cancers, colorectal cancer, and head and neck cancers.

EXAMPLE 1 Production Method of rScl1 Hybrid Proteins

rScl proteins are produced in E. coli, and expressed either intracellularly or in the periplasmic space. The extracellular fraction is recovered from culture supernatant following precipitation with ammonium sulphate. Production yields vary between rScl1 constructs, ranging from 1-20 milligrams of protein per liter of culture.

Sequences encoding the rScl.hybrid1 and rScl.hybrid2 proteins were cloned and expressed in an E. coli Strep-tag II system. The hybrid proteins were recovered from E. coli following incubation in a high-sucrose buffer or via cell lysis, and then purified by affinity chromatography, using StrepTactin Sepharose. The proteins were then subjected to dialysis to desired exchange buffers and stored at −20° C.

EXAMPLE 2 Construction of Recombinant rScl Hybrid Proteins 1 and 2

Recombinant rScl.hybrid1 and rScl.hybrid2 constructs were generated by several sequential manipulations. Both proteins were derived from rScl proteins rScl1.1, rScl1.41 and rScl2.28. Construct rScl.hybrid1 harbors a CL-domain that contains tandem integrin-binding motifs ((GLPGER)₂₎ recloned from the CL-domain of rScl1.41 into the biologically inert CL-domain of rScl2.28, and the V-domain of rScl1.1 with ECM (EDA/cFn, EDB/cFn, and TNC) binding domain.

The rScl.hybrid2 protein is comprised of the CL-domain of rScl.hybrid1 and the rScl2.28 V-domain modified to contain the ECM-binding domain.

EXAMPLE 3 Binding of rScl Proteins to rEDA, rEDB and TNC

Recombinant Scl1 proteins were tested for binding to recombinant EDA and EDB (FIG. 2A), as well as Tenascin-C (FIG. 2B). rScl proteins were immobilized onto Strep-Tactin-coated microplate wells and incubated with rEDA/rEDB or TNC. Primary anti-rEDB or TNC antibodies and HRP-conjugated secondary antibodies were used for ligand detection. The shown graphic bars indicate mean OD_(415nm) normalized against BSA controls. FIG. 2A shows that both rScl1.1 protein derived from a natural sequence and the engineered rScl.hybrid1 recognize recombinant EDB to a comparable level as recombinant EDA. FIG. 2B shows ELISA binding data between rScl1 proteins and TNC extracted from glioblastoma cancer cells.

Although the various embodiments have been described in detail with particular reference to certain aspects thereof, it should be understood that the invention is capable of other embodiments and its details are capable of modifications in various obvious respects. As is readily apparent to those skilled in the art, variations and modifications can be effected while remaining within the spirit and scope of the invention. Accordingly, the foregoing disclosure, description, and figures are for illustrative purposes only and do not in any way limit the invention, which is defined only by the claims.

Amino acid sequence of rScl.hybrid1 SEQ ID NO: 1 EVSSTTMTSSQRESKIKEIEESLKKYPEVSNEKFWERKWYGTYFKEEDFQ KELKDFTEKRLKEILDLIGPTGLAGEKGEAGPQGEKGLPGLTGLPGLPGE RGPRGPKGDRGETGAQGPAGEKGEAGPQGEKGLPGLTGLPGLPGERGPRG PKGDRGETGAQGPAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEA GAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGK DGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDG LPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPAPKTPEVPQKPD TAPWSHPQFEK Amino acid sequence of rScl.hybrid2 SEQ ID NO: 2 DEQEEKAKVRTELIQELAQKYPEVSNEKFWERKWYGTYFKFLTYLQEREQ AENSWRKRLLKGIQDHALDGLAGEKGEAGPQGEKGLPGLTGLPGLPGERG PRGPKGDRGETGAQGPAGEKGEAGPQGEKGLPGLTGLPGLPGERGPRGPK GDRGETGAQGPAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGA QGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDG ERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLP GKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPAPKTPEVPQKPDTA PWSHPQFEK Amino acid sequence of rScl1.1 SEQ ID NO: 3 EVSSTTMTSSQRESKIKFIEESLKKYPEVSNEKFWERKWYGTYFKEEDFQ KFLKDFTEKRLKEILDLIGKSGIKGDRGETGPAGPAGPQGKTGERGAQGP KGDRGEQGIQGKAGEKGERGEKGDKGETGERGEKGEAGIQGPQGEAGKDG APGKDGAPGEKGEKGDRGETGAQGPVGPQGEKGETGAQGPAGPQGEAGKP GEQGPAGPQGEAGQPGEKAPEKSPEGEAGQPGEKAPEKSKEVTPAAEKPW SHPQFEK Amino acid sequence of rScl2.28 SEQ ID NO: 4 DEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQ EREQAENSWRKRLLKGIQDHALDGQDGRNGERGEQGPTGPTGPAGPRGLQ GLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGP AGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGEPGTQGAKG DRGETGPVGPRGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKD GQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGK DGKDGQPGKPAPKTPEVPQKPDTAPWSHPQFEK Amino acid sequence of rScl1.41 SEQ ID NO: 5 EDSETATARTKLLEKLTELRSQSQDRVPQTSDITQAYTLWGTSYDSVELY KYLQQIEEYLQKQKYHEEQWKKEITDGLKSGALRGEKGEAGPQGEKGLPG LTGLPGLPGERGPRGPKGDRGETGAQGPVGPQGEKGEAGTPGKDGLRGPQ GDPGAPGKDGAPGEKGDRGETGAQGPVGPQGEKGEAGTPGKDGAPGEKGE KGDRGETGATGAQGPQGEAGKDGAQGPVGPQGEKGETGAQGPAGPQGEKG ETGAQGPAGPQGEAGQPGEKAPEKSPEVTPTPEMPEQPGEQAPEKSKFVT PAPEKPWSHPQFEK Integrin recognition sequence SEQ ID NO: 6 GLPGER 

1. A recombinant collagen-like protein comprising a binding domain having binding capacity for both extra domain A and extra domain B-containing variants of cellular fibronectin.
 2. The recombinant collagen-like protein of claim 1, wherein the protein is a prokaryotic collagen-like protein.
 3. The recombinant collagen-like protein of claim 1, wherein the protein is a streptococcal collagen-like protein from a Group A Streptococcus.
 4. The recombinant collagen-like protein of claim 3, wherein the Group A Streptococcus is Streptococcus pyogenes.
 5. The recombinant collagen-like protein of claim 4, wherein the streptococcal collagen-like protein is derived from Scl1 or Scl2 of Streptococcus pyogenes or a combination thereof.
 6. The recombinant collagen-like protein of claim 1, wherein the protein further comprises binding capacity for Tenascin-C.
 7. The recombinant collagen-like protein of claim 1, wherein the protein further recognizes collagen-binding integrin receptors α₂β₁ and α₁₁β₁.
 8. The recombinant collagen-like protein of claim 1, wherein the protein comprises two copies of an integrin recognition sequence GLPGER.
 9. The recombinant collagen-like protein of claim 8, wherein the protein further comprises the variable domain of the rScl1 sequence of SEQ ID NO:
 3. 10. An expression vector comprising a nucleotide sequence encoding the recombinant collagen-like protein of claim
 1. 11. A host cell comprising and expressing the expression vector of claim
 10. 12. A recombinant collagen-like protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:
 2. 13. A pharmaceutical composition comprising a recombinant collagen-like protein comprising a binding domain having binding capacity for both extra domain A and extra domain B-containing variants of cellular fibronectin; and a pharmaceutically acceptable carrier.
 14. The composition of claim 13, wherein the carrier is a nanoparticle.
 15. The composition of claim 14, wherein the nanoparticle is coated with the collagen-like protein.
 16. A method of treating cancer comprising administering a recombinant collagen-like protein comprising a binding domain having binding capacity for both extra domain A and extra domain B-containing variants of cellular fibronectin.
 17. The method of claim 16, wherein the recombinant collagen-like protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO:
 2. 18. The method of claim 16, wherein the cancer is selected from a group consisting of a tumor or a metastatic cancer.
 19. The method of claim 16, wherein the cancer is selected from a group consisting of breast cancer, prostate cancer, melanoma, gastric cancer, colorectal cancer, and head and neck cancer.
 20. A method of preparing a collagen-like protein comprising a binding domain having the capacity to bind to both extra domain A and extra domain B-containing variants of cellular fibronectin comprising the steps of combining: (i) two copies of an integrin recognition sequence of SEQ ID NO: 6 inserted into the collagen-like (CL) domain of a protein of SEQ ID NO: 4; and (ii) the variable (V) domain from a protein of SEQ ID NO:
 3. 